Although steady state kinetics of acetylcholinesterase catalyzed reactions have received considerable attention, not much study has been devoted to the transient reaction kinetics or equilibrium interactions between enzyme and effector ligands. Reasons for the discrepancy in available information are straightforward: (1) General unavailability of purified enzyme in quantities comparable to substrate or ligand concentration; and, (2) The difficulties in obtaining substrates and effectors containing signals directly related to the mode of interaction. Both of these difficulties are obviated by steady state kinetic studies since minute amounts of enzyme are required, and effector "interaction constants" can be derived from their concentration-dependent effect on activity. Continuing and heightening interest in the mechanism of synaptic transmission and its regulation prompts more detailed investigations into cholinergic effector anacetylcholine interactions with both the acetylcholine-receptor protein and with the enzyme Acetylcholinesterase (AChE). Although the receptor and hydrolytic enzyme proteins have been demonstrated to be distinct (different molecules), the relative potency of effectors and their effects on acetylcholine-site interactions has been found to be remarkably similar. An essential feature of this proposal is to develop methods for monitoring ligand-protein interactions and reactions directly, and to apply these methods to studies of enzyme-ligand interaction; and subsequently to receptor-ligand interactions and the interactions of ligands with vesicle- and membrane-bound proteins. The methods of monitoring interactions, if successfully developed, should also be appropriate for examining such interactions in intact cells and tissues. This laboratory has contributed substantially to the successful development of such methods for both monomeric hydrolytic enzymes and for more complex allosteric (regulatory) glycolytic enzymes. The proposal outlined represents a logical next stage in complexity.